Homologous recombination between DNA sequences placed on the same plasmid is well known to the skilled artisan (Weber and Weissmann, Nucl. Acid. Res. (1983) 11, 5661-5669). EP 252666 B1 (Novo Nordisk A/S), WO95/22625 A1 (Affymax Technologies N.V.) and WO9101087 discloses in vivo recombination of genes placed on the same plasmid.
EP 449923 B1 (Setratech) discloses a process of intergenic recombination in vivo of partially homologous DNA sequences. The recombination takes place in cells of which the enzymatic mismatch repair system is defective.
J. Biotechnol. (1991) 19, 221-240 discloses a process for inactivating a gene in the genome of B. Amyloliquefaciens. The process involves integration and excision of a pE194 mutant.
In Chemical Abstracts 118:161925 (1993) the minimal sequence length of homology necessary for recombination is determined. The method of determination involves integration of a pE194 derivative carrying a gluconase gene.
In J. Bacteriol. (1982) 152, 524-526 a plasmid pBD9, which comprises the two plasmids from Staphylococcus aureus, pE194 and pUB110, is disclosed. Other plasmids based on pE194 or pUB110 are disclosed in Molecular and General Genetics (1988), 213, 465-70, Molecular and General Genetics (1984), 195, 374-7, Plasmid (1981), 6, 67-77 and Genetika, (1986) 22, 2750-7.
In Yichuan Xuebao (1993), 20, 272-8 (see Chemical Abstracts 120:1670 (1994)), Chen et. Al. disclose the plasmid pNW102, a pE194 derivative, in which the thermostable .alpha.-amylase gene from Bacillus licheniformis is inserted. pNW102 was transformed into the Bacillus subtilis strain BF 7658, followed by incubation at non-permissive temperature, allowing homologous recombination between the .alpha.-amylase genes of pNW102 and BF 7658. The .alpha.-amylase produced of the recombinant strains shows the same characteristics as the .alpha.-amylase from Bacillus licheniformis.
However, in the related art there is no indication of that DNA sequences can be recombined in vivo by repeating integration and cross-out in order to obtain a gene encoding a polypeptide with improved characteristics, such as increased stability, improved specificity or higher activity.
Accordingly, a problem of the invention is to provide an improved in vivo process of generating new DNA sequences.